Translation of abstract (English)

Somatic gene therapy using retroviral vectors could be used successfully for the treatment of monogenetic inherited diseases. However, with increased efficiency vector-induced severe side effects have already been reported in 3 clinical gene therapy trials. There, integration site (IS) analyses allowed the precise identification and sequencing of the IS locus in the affected cells. In all reported severe adverse events the integrants were found in or close to proto-oncogenes that led to an overexpression of these genes and to malignant cell transformation. Finally 2 of the 7 diseased patients died of the consequences. Thus, comprehensive analyses of the vector integrations and their effects on cellular biological processes are of prime importance and can help to assess the potential risk at an early stage on the molecular level. In this thesis, IS identification was carried out by using the linear amplification mediated PCR (LAM-PCR) method. To determine the genomic access to viral IS in the host genome, a mathematical model was developed, that enabled the calculation of the genomic accessibility to viral IS a priori in dependence of the used restriction enzymes. Moreover, we could develop a non-restrictive (nr) LAM-PCR without the restrictive application of restricition enzymes conventionally needed in IS analyses. Our comparative IS analysis combined with sanger sequencing was carried out in the scope of this thesis with 5 clinical (2547 IS) and 3 preclinical (1316 IS) gammaretroviral studies, showing impressive correlations between the studies. Thus, more than 70% of the integrations were found to be located in gene coding regions with an accumulation around the transcription start site (TSS, 23%-39%). Furthermore, we found the proto-oncogenes MDS1-EVI1 (108 IS), PRDM16 (37 IS), LMO2 (13 IS), CCND2 (12 IS) and SETBP1 (10 IS) as most common favored integration loci. A further focus presented the clonality and pharmacokinetic analyses of samples from 9 patients from two X-CGD, one ADA-SCID and one WAS gammaretroviral clinical gene therapy study over a period of 5 years after therapy. For all analyzed studies, (nr) LAM-PCR followed by pyrosequencing (GS FLX, > 20000 IS) also observed a non-random IS distribution with favored integrations in gene coding regions (47%-72%) and an accumulation around the TSS (8%-16%). The results further also revealed the detection of favored integrations in or close to the proto-oncogenes MDS1-EVI1 (289 IS), PRDM16 (104 IS), LMO2 (52 IS), SETBP1 (34 IS) and CCND2 (33 IS). Using LAM-PCR „Sequence Count” analyses a vector-induced in vivo selection of individual MDS1 clones was observed in 3 out of 4 treated X-CGD patients. Reverse Transcriptase (RT)-PCR analyses of the gene expression in one X-CGD patient showed an overexpression of the affected genes MDS1-EVI1 and STAT3. For all further patients of the analyzed clinical gene therapy studies the hematopoietic repopulation stayed polyclonal until the latest time point analyzed. However, for the WAS gene therapy trial we could also detect an in vivo selection of one CCND2 and one MDS1 clone by „Sequence Count” analysis and qPCR. The clonality analyses using LAM-PCR/nrLAM-PCR in combination with next generation sequencing (pyrosequencing, GS FLX) led to the generation of a unique data collection of > 20000 total IS from clinical patient material. Our analyses revealed that conventional retroviral vectors with an active LTR have a really significant influence on the cellular gene expression. Long-term studies will help to clarify the biological consequences of the retro- and lentiviral selfinactivating (SIN) LTR vector systems on the fate of the transduced cell.